Abstract 2485: RNA editing by APOBEC3s in lung cancer tumors largely occurs in infiltrating immune cells

Abstract

Abstract Introduction: APOBEC3 enzymes contribute significantly to DNA mutagenesis in cancer. Our recent studies show that these enzymes are also capable of converting C bases at specific positions (sites) of RNAs to U. By examining primary non-small cell lung cancer (NSCLC) tumors of The Cancer Genome Atlas (TCGA) project, we have demonstrated that such APOBEC3-mediated C-to-U RNA editing occurs in lung cancer tumors. While we could detect editing events in the tumors, their cellular origin remains unclear. Here, we investigated if the editing occurs in cancerous or stromal cells of lung cancer tumors. Methods: Immune-related features of the TCGA lung cancer tumors (n = 1,009) were obtained from various TCGA studies to examine their association with APOBEC3-mediated RNA editing that we had determined previously. Freshly resected late-stage NSCLC tumors of five patients were mechanically and enzymatically dissociated into single cells using a cocktail of collagenase, elastase, and DNAse enzymes. Antibodies conjugated to magnetic beads were then used to isolate cancerous epithelial (EpCAM+) and stromal pan-immune (EpCAM-/CD45+) and macrophage/monocyte (EpCAM-/CD14+) subsets of cells from the single-cell suspensions. RNA (depleted of rRNA) and genomic DNA of the cell isolations were subjected to total RNA and whole exome sequencing. Sequencing data was examined to identify C-to-U RNA editing at 5,208 known APOBEC3-mediated editing sites. Results: TCGA lung tumors with higher RNA editing levels had stronger tumoral immune response. Particularly, they had about 25% more infiltrating leukocytes, CD8 T cell fraction, and immune cytolytic activity compared to tumors with low editing level (P < 0.01 in all t tests). As expected, expression of genes that are markers of epithelial (such as EPCAM and MUC1), pan-immune (CD45, ITGAL), and macrophage/monocyte (CD14, FCGR3A) cells were dramatically higher among the corresponding cell subsets that were isolated from lung tumors. Whereas expression of APOBEC3B and F were similar among the three subsets, the expression of APOBEC3A, C, D, G and H were 2-4-fold higher in pan-immune and macrophage/monocyte compared to epithelial cells. Concordantly, while editing events were seen in the epithelial cells at average 7% of examined sites, editing was more frequent in the pan-immune and macrophage/monocyte cells (17% and 22%, respectively). Conclusions: RNA editing by APOBEC3s reflects a heightened immune response in lung cancer. A large proportion of RNA editing by APOBEC3s in tumors occurs in immune cells such as macrophages. Further investigations are needed to understand RNA editing in various other immune cells, as well as the relevance of RNA editing of specific genes in cancer and immune cell biology. Citation Format: Mariko Asaoka, Eric Kannisto, Takashi Ishikawa, Sai Yendamuri, Santosh Kumar Patnaik. RNA editing by APOBEC3s in lung cancer tumors largely occurs in infiltrating immune cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2485.